Thrombophilia PDF

Paul Ellery, PhD [email protected]  Intravascular occlusion due to inappropriate clotting  Single largest cause of death in the world  Venous  Arterial 2  Most commonly occurs in the legs  Thrombophlebitis  Thrombosis in...

Paul Ellery, PhD [email protected]  Intravascular occlusion due to inappropriate clotting  Single largest cause of death in the world  Venous  Arterial 2  Most commonly occurs in the legs  Thrombophlebitis  Thrombosis in the superficial veins  Inflammation or stasis  Self-resolving  Deep vein thrombosis  Most common site is in the deep veins of the legs  Pulmonary embolism is a serious complication  Other sites  Arm/shoulder, renal vein, splanchnic veins (veins of the abdominal organs), retinal veins 3 4 DVT PE Direction of Blood Flow 7  Thrombi commonly form around the valves of the leg veins  VT initiates after endothelial cell activation, → fibrin formation at the endothelial surface  Fibrin traps RBCs and platelets  “Red thrombus”  Clot can break up into pieces, travel through the heart, and lodge in the pulmonary circulation 8 Direction of Blood Flow 9  Not well understood  Virchow’s triad  Described in mid-1800’s  VT due to at least one of three causes  Increase in blood coagulability  Changes in the vessel wall  Blood stasis 10  Mechanisms of endothelial activation  Inflammation  Stasis at the back of the valve → hypoxia → EC activation  Activated ECs  Express TF  Express P-selectin  Allows leukocytes to bind to the endothelium  Release vWF  Allows platelets and leukocytes to bind to the endothelium 11  Stasis  Accumulation of prothrombotic factors  Hypoxia in leukocytes, platelets, (and ECs)  Expression of TF in monocytes  Production of TF-bearing microparticles  Anticoagulant function of the endothelium is decreased in large vessels 12  Symptomatic patient  Presence of risk factors  D-dimer  Elevated in VTE  Not diagnostic, but can be used to exclude VTE  Imaging (ultrasound etc) 13  ? Thrombolytic therapy  Initially, heparin  Usually LMWH  UFH only in certain patient populations  Then warfarin or other orally administered anticoagulant  Continues for 3-6 months, to years 14 15 16  Hospitalisation  Similar incidence when hospitalised for medical illness vs surgery  Nursing home  Lupus anticoagulant  Active cancer  Trauma/fracture  Pregnancy  Oral contraceptives/oestrogen  Obesity  Long-haul journeys  Genetic predisposition (Hereditary Thrombophilia) 17 21  Congenital risk factors for the development of thrombosis  Presence doesn’t confer disease  Affected individuals usually present with  Venous thromboembolism (VTE) at a young age  Recurrent VTE  Family history of VTE 22  Reduced levels of the naturally occurring anticoagulants:  Antithrombin (AT)  Protein C (PC)  Protein S (PS)  AT and PC have type I and type II deficiencies  Type I – decreased antigenic levels of functional protein  Type II – normal antigenic levels of protein with decreased function  Factor V Leiden (FVL)  Prothrombin Gene Mutation (G20210A) 23  AT inhibits serine proteases  Main targets are IIa (thrombin), Xa, IXa, XIa, XIIa.  Activity is greatly enhanced by heparin/heparan sulphate  Contains two major functional regions  Reactive site  Heparin/heparan binding 24  Both functional and antigenic assays are available for the measurement of AT  Functional – heparin co-factor assay  Patient plasma is mixed with heparin and thrombin  Residual thrombin activity is determined with the addition of a thrombin-specific chromogenic substrate  Clot-based assays are also available  Should be used to measure AT levels  Antigenic – ELISA, latex-agglutination  AT reference range is relatively narrow 25  Congenital AT deficiency is rare  0.02% in the general population  0.5-1% in the thrombotic population  Type II more common than Type I  Affected individuals experience their first thrombotic episode in the first 25 years of life  Heterozygous deficiency confers a 10-20 fold increased thrombotic risk  Homozygous deficiency is incompatible with life  Except for mutations in the heparin-binding site 26  Contains Protein C (PC), Protein S (PS), Thrombomodulin (TM), and the Endothelial Protein C Receptor (EPCR)  PC and PS are vitamin-K dependent proteins produced by the liver  TM is expressed on the EC surface  Binds “non-clot bound” thrombin  Changes thrombin’s substrate specificity  EPCR is expressed on the endothelial surface, binds PC 27  Protein C/S System Thr PC TM EPCR  Protein C/S System FVa/ FVIIIa A TM Thr P PS C EPCR  Protein C/S System FVa/ FVIIIa A TM Thr PS P EPCR C  Measured by activity and antigenic assay  Activity assays  Should be used to measure PC  Chromogenic or clot-based (aPTT)  Both use Protac, which activates Protein C  In the clot-based assay, normal plasma has a prolonged time (< 100s), whereas PC ↓ plasma has a normal time (30-40s)  Chromogenic assays do not detect PC that has abnormal PL or Ca2+ binding, but are preferred over clot-based assays  Clot-based assays may underestimate PC activity in the presence of FVL, ↑ FVIII, hyperlipidaemia, and may give unreliable results in the presence of lupus anticoagulants 31   Congenital PC deficiency  0.3% of the general population  ~3-5% of the thrombosis population  Type I more common than type II  Incomplete penetrance  Heterozygous deficiency confers a 10-15 fold increase in thrombotic risk  Homozygous deficiency → purpura fulminans and DIC in newborns Image taken from http://fn.bmj.com/content/fetalneonatal/early/2015/03/17/archdischild-2015-308238/F1.large.jpg 32  Two pools of PS are present in plasma  ~60% is bound to C4bp. No co-factor function.  ~40% circulates unbound  Free vs total PS  Assays  Total PS antigenic assay  Free PS antigenic assay  PS functional (activity) assay  Low levels found in the functional assay should be confirmed with free PS antigenic assay 33  Three types of deficiency  Type I  Normal PS protein is produced at a reduced level  Decreased antigenic and functional PS  Type II  Normal levels of PS with decreased functional activity  Normal antigenic levels, decreased functional levels  Type III  Decreased free PS antigen, normal total PS antigen 34  Congenital PS deficiency occurs in:  0.2% of the general population  ~10% of the thrombophilic population  Incomplete penetrance  Heterozygous deficiency confers a 10-15 fold increase in thrombotic risk  Homozygous deficiency → purpura fulminans and DIC in newborns 35  aka the Prothrombin G20210A mutation  2-3% of the general population are heterozygous  ~10-45% of thrombophilia population are heterozygous  Almost exclusively in Caucasians  Increases thrombotic risk by ~2-fold 36  Mutation in the 3’ UTR of the F2 gene  Increases the stability of the F2 mRNA  Heterozygotes have elevated plasma Prothrombin relative to homozygous WT (~30% increase)  Elevated Prothrombin → increased thrombin generation  Can only be identified by molecular testing 37  The most common thrombophilia  ~5% of the general population are heterozygous, 100 Prothrombin Gene Mutation, heterozygous 3 PGM, homozygous ??? PGM, heterozygous + OCP 16 Protein C deficiency, heterozygous 7 Protein C deficiency, homozygous Severe thrombosis at birth Protein S deficiency, heterozygous 6 Protein S deficiency, homozygous Severe thrombosis at birth Antithrombin deficiency, heterozygous 5 Antithrombin deficiency, homozygous Thought to be lethal prior to birth 54  Should not be performed until at least 3 months after an acute thrombotic episode  Functional assays should not be performed while the individual is on anticoagulant therapy  Assays for AT, PC, and PS should be repeated on more than one occasion  Molecular testing can be performed at any time  Family studies 55 DNA vWF EPCR TM Overlay Valve Sinus Valve Lumen 56 58  Arterial Clot Formation Change in blood flow due to plaque causes “eddy’s” to form that can activate platelets Atherosclerotic plaque formation Formation of clot on the leeward side of the plaque mostly platelets/fibrin  Arterial Clot Formation Atherosclerotic plaque formation Plaque rupture exposes macrophages that express high levels of TF on their surfaces  Arterial Clot Formation TF TF TF Plaque rupture exposes macrophages that Clotexpress highoflevels comprised of TF macrophages, on their surfaces platelets and fibrin Not usually associated with embolism, more likely to be a blockage at the site of plaque rupture Image taken from Libby and Theroux. Circulation (2005) 111: 3481-3488 62  Age  Gender  Family history  Blood lipids  Obesity  Hypertension  Cigarette Smoking  Diabetes  Diet  Exercise  Homocysteine 63  No well defined links between coagulation factors and risk of arterial thrombosis  ? Plasma Fibrinogen  Affected individuals are treated with heparin and warfarin/new orally-administered anticoagulants, platelet inhibitors 64 65  Thrombocytopaenia after the administration of heparin, due to thrombus formation  Occurs in ~ 1 in 5000 hospitalised patients  More common with UFH than LMWH administration (incidence rate: 1-3% vs ~0.2%)  Begins 5-10 days after heparin exposure  Exceptions to the rule – rapid-onset HIT, delayed-onset HIT 66 67 68  50% or more decrease in platelet count, or thrombus development, within 5-10 days of initiation of heparin therapy  Decrease is relative to the highest platelet count after the initiation of heparin  Count rapidly decreases (1-3 days)  Monitoring of platelet count recommended in populations where risk of developing HIT is increased 69 70  Platelet Count  Immunoassays to detect antibodies against heparin-PF4 complexes  Excellent negative predictive value  Poor positive predictive value  Assays identify clinically-insignificant antibodies against heparin-PF4 complexes  Functional platelet studies  More specific than immunoassays for the detection of clinically significant antibodies  Serotonin Release Assay  Gold standard, but not routinely performed  Aggregometry  Flow cytometry 71 72  Stop heparin therapy  Transfer to direct thrombin/Xa inhibitor  Can not use warfarin until platelet count has increased 73 74  Microangiopathic haemolytic anaemia w/ schistocytes and thrombocytopenia  Caused by a deficiency in ADAMTS13  Congenital or acquired  More common in adults  ADAMTS13  A Disintegrin and Metalloprotease with a Thrombospondin Type I motif, member 13  Cleaves ultra-large von Willebrand Factor (UL-VWF) multimers 75 76 77 78  Anaemia and thrombocytopenia in the absence of leukopenia  Schistocytes and polychromatophilic RBCs  Increased LDH, bilirubin, decreased haptoglobin  Negative DAT  N PT and aPTT  Increased UL-VWF  ADAMTS13 assays  < 5% activity 79  Plasmapheresis  Cryoprecipitate-depleted plasma  Should be started ASAP, because TTP can fatal and is easily treatable 80  Briefly describe the 5 major causes of hereditary thrombophilia and their measurement in the laboratory  Briefly describe the APC resistance test and how it can be modified to specifically measure FV Leiden or other causes of APC resistance  Briefly describe the pathogenesis, diagnosis, and treatment of heparin-induced thrombocytopenia  Briefly describe the pathogenesis, diagnosis, and treatment of thrombotic thrombocytopenic purpura 81  Dacie and Lewis. 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